PROJECT SUMMARY The mammary epithelium is composed of diverse cell populations that are responsible for regulating key processes in mammary gland development, especially lactation. Alveolar progenitor (AP) cells are partially differentiated stem cells that produce alveolar/milk-producing cells and these cells have recently been implicated as cells of origin in breast cancer. Through analysis of published single cell RNA-seq data, integrin ?4, a protein known to function primarily in basal mammary epithelial cells to maintain structural integrity of the gland, was found to be expressed in the AP population. This unexpected result suggests that ?4 plays a novel role in the AP population and may regulate dynamic processes within the developing mammary gland. This goal of this proposal is to understand the functional role of ?4 in the AP cells of the nulliparous and lactating mammary gland and elucidate the mechanism by which ?4 regulates this population. Preliminary studies have revealed that ?4 expression is necessary to promote progenitor potential of the AP cells and identified LacdiNAc, a novel glycosylation modification, on ?4 that we hypothesize is essential for its localization in lipid rafts that promotes its function in the AP population. Using an AP cell culture model as well as a Cre-lox mouse model to conditionally knock out ?4 from the AP population, the function of ?4 in the AP population will be assessed in vitro and in the virgin and lactating mammary gland. Also, glycomics analyses and biochemical approaches will identify novel glycans on ?4 and help in understanding how LacdiNAc affects function of ?4 in the AP population. This approach will further our understanding of the novel role of ?4 in the AP population and a new mechanism involving LacdiNAc-?4 localization to lipid rafts to regulate alveolar differentiation. These studies have to potential to define novel mechanisms that regulate the AP population during normal mammary gland development as well as breast cancer progression.